Controlling Gram-negative bacteria with natural growth promoters in broiler production

For modern poultry production, bacterial contamination of the gastro-intestinal tract, especially Gram-negative bacteria contamination, is a major problem.

Many approaches have been made to protect poultry against contamination with Gram-negative bacteria and to tackle infection when it occurs, including use of antibiotics and other substances. One of the longest-established methods used is the acidification of the feed and the gastrointestinal tract.

Organic acids have the ability to modulate the intestinal microflora, enhancing the growth of beneficial bacteria such as lactobacilli, while reducing the number of pathogenic Gram-negative bacteria. In the modern chicken industry, combinations of various organic acids are used because of the broader spectrum of activity offered.

The main function of the gut is the uptake of nutrients. For this to happen at an optimum level, a balance in the intestinal microflora is needed to ensure not only good digestion and absorption of nutrients but also to protect the chicken from harmful bacteria via competitive exclusion. Substances able to alter the intestinal microflora include organic acids and phytochemical substances, among others. They have an antibacterial effect and are able to target only the pathogenic bacteria while sparing the beneficial bacteria or even increasing their numbers.

Organic acids

When people talk about organic acids in the poultry industry, they are usually referring to their pH-reducing effect. In addition to reducing the pH of the environment when in dissociated form, organic acids in undissociated form also have an indirect antimicrobial action.

Every organic acid possesses a so-called pKa value (Table 1), which shows how much of the acid will dissociate and how much will remain in undissociated form, depending on the pH on the environment. With this pKa value, it is possible to determine if an acid will be in the dissociated or undissociated form in a given pH environment so that the most suitable product can be chosen for use in the poultry feed or water.

Based on the table, propionic acid has a pKa value of 4.88. This means that, in an environment with a pH of 4.88, half of the propionic acid will be found in dissociated form and the other half in undissociated form.

Acids in dissociated form act as pH reducing agents of the environment by releasing H+ ions. In undissociated form, acids are able to enter the bacterial wall where they will find a near neutral environment in the cytoplasm of the bacteria.

Dissociation takes place in this environment, reducing the pH while disrupting the DNA, protein synthesis and internal membranes. This causes the bacteria to use up significant amounts of energy regulating the intracellular pH. The anions of organic acids trapped inside the bacterial cell act as toxic compounds that inhibit the metabolic reactions and stop the growth of the bacteria.

Phytochemical -- cinnamaldehyde

Cinnamaldehyde is the organic compound that gives cinnamon its flavor and odor. It is derived naturally from the bark of cinnamon trees and other species of the genus Cinnamomum. It is used as a flavoring agent because of its pleasant smell and taste, as an agrichemical because of its anti-fungal properties, as an anti-corrosive agent and as an antimicrobial.

The antimicrobial action of the cinnamaldehyde comes from its ability to block the formation of the FtsZ proteins which play an important role in the cell division process.

Normally, FtsZ proteins are present in the cell, forming filaments which migrate to the middle of the cell where cell division takes place. Here, they form a ring, which eventually splits the cell, causing the cell to divide and multiply. If the FtsZ protein formation is blocked by cinnamaldehyde, cell division cannot take place.

It has also been shown by J. Michiels et al., 2009 that cinnamaldehyde has a selective action against pathogenic bacteria but does not affect beneficial bacteria (Table 2).

Combining different organic acids enhances activity against a broader range of Gram-negative bacteria, and adding cinnamaldehyde to the mixture can further enhance the antimicrobial effect of the product and broadens its antimicrobial spectrum.

Fighting Gram-negative bacteria

Gram-negative bacteria have an extra outer membrane; however, that provides the bacteria with an inherent resistance against antimicrobial substances. This membrane consists of lipopolysaccharides, protects bacteria from the action of bile salts and digestive enzymes and provides special resistance to hydrophobic antibiotics and detergents.

To fight successfully against Gram-negative bacteria, this membrane must be damaged. Permeabilizing substances are able to permeate this outer membrane, thereby facilitating the entry of antimicrobial substances into the bacterial cell.

In vitro trials have been conducted by Biomin to show the efficacy of permeabilizers in boosting the antimicrobial effect of organic acids and cinnamaldehyde.

A control group with no antimicrobial substance added was compared to three other groups: an antimicrobial mixture (organic acids + cinnamaldehyde), a permeabilizer alone, and an antimicrobial mixture with permeabilizer. The trial was conducted using Salmonella typhimurium and Escherichia coli strains.

The results (Figure 1, Figure 2) showed that the permeabilizer alone had no effect on bacterial growth inhibition. The antimicrobial mixture had an inhibiting effect in the first hours of the trial, while the combination of antimicrobial mixture and permeabilizer totally inhibited bacterial growth during the whole trial period. These results show that the permeabilizer has the potential of boosting the effect of the organic acids and cinnamaldehyde, inhibiting the growth of Gram-negative bacteria.

Because of the growing demand for poultry meat, there is a need to create optimum feeding conditions for broilers to perform at the highest possible level and combining organic acids with phytochemicals may offer new possibilities in the fight against Gram-negative bacteria.

Because the outer membrane of Gram-negative bacteria makes it difficult for some antimicrobial alone to work against them, permeabilizing substances are needed to boost the effect of these antimicrobial substances. Three active ingredients -- an organic acid blend, cinnamaldehyde and a permeabilizer -- offer an approach to pathogen control, thus contributing to optimal performance for poultry performance.